The demand of acquiring large amounts of a specific segment of DNA efficiently for different purposes is booming in recent years. Among the entire existing DNA sequencing techniques, Polymerase Chain Reactions (PCR) is one of the most economical and straightforward techniques amplifying billion copies of targeted DNA segments in short period of time. The applications of PCR technique are broadly adopted, such as selective DNA isolation for genetic identification, forensic analysis for analyzing ancient DNA in archeology, medical applications for genetic testing and tissue typing, fast and specific diagnosis of infectious diseases for hospitals and research institutes, inspection of environmental hazards for food safety, genetic identification for investigating criminals, and so on. For PCR technique, only small amount of DNA samples are required from blood or tissues. By utilizing a fluorescent probe into the nucleic acids solutions, the amplified DNA segments could be detected through the help of fluorescent molecules.
To simultaneously detect and analyze the presence of targeted nucleic acids in a batch of biological samples, fluorescent dyes detection technique is usually applied. After the light source at specific wavelength illuminates on the targeted nucleic acids, the DNA-binding fluorescent probes of the nucleic acids will react and enable fluorescent signals to be emitted. The fluorescent signal is an indication of the existence of the targeted nucleic acids. This technique has been employed for the novel PCR technique, which is called real time quantitative PCR or qPCR. qPCR is the early-phase PCR detection with higher sensitivity and better precision than the conventional PCR technique which is an end-point PCR detection. An optical device is essential to detect the fluorescent light emitted from the specific nucleic acids segments for qPCR technique. The optical device has to provide a light source to excite the fluorescent probe at their specific wavelengths, and in the meanwhile, it detects the fluorescent signals emitted from the fluorescent probe.
There are a large number of fluorescent detection instruments known in the art that are employed to image fluorescence signals, but the bulky size and weight of such instruments are huge and the signal-to-noise ratio (SNR) thereof cannot meet the practical requirements. On the other hand, another problem that all fluorescence detection instruments have in common is the huge intensity difference between the excitation light and the fluorescence light signal. If at least one fluorescent probe is involved in the multiplexing, to isolate different excitation and fluorescence signals become a very important task. In general, different excitation filters are required when multiple fluorescent probes are present in a sample. Each excitation filter is able to transfer at least one excitation light at specific wavelengths from the light source to the assembly of multiple individual sites. A large number of expensive optical devices have to be employed with respect to different excitation filters, respectively. While a sample including multi fluorescent probes, the sample has to be placed in several different sites for detecting sequentially. It is not convenient and the detection result will be influenced due to the temperature and reaction time changes.
In light with the requirements and the issues addressed above, there is a need of providing an improved fluorescence detection instrument with high signal-to-noise ratio for multiplexing qPCR application.